Many cancers are characterized by disruptions in cellular signaling pathways that lead to uncontrolled growth and proliferation of cancerous cells. Receptor tyrosine kinases (RTKs) play a pivotal role in these signaling pathways, transmitting extracellular molecular signals into the cytoplasm and/or nucleus of a cell. Cells of virtually all tissue types express transmembrane receptor molecules with intrinsic tyrosine kinase activity through which various growth and differentiation factors mediate a range of biological effects (reviewed in Aaronson, Science 254: 1146–52 (1991)). RTKs share a similar architecture, having an intracellular catalytic domain, a hydrophobic transmembrane domain, and an extracellular ligand-binding domain. The binding of ligand to the extracellular portion is believed to promote dimerization, resulting in trans-phosphorylation and activation of the intracellular tyrosine kinase domain (see Schlessinger et al., Neuron 9:383–391 (1992)).
Biological relationships between various human malignancies and disruptions in growth factor-RTK signal pathways are known to exist. For example, overexpression of EGFR-family receptors is frequently observed in a variety of aggressive human epithelial carcinomas, such as those of the breast, bladder, lung and stomach (see, e.g., Neal et al., Lancet 1: 366–68 (1985); Sainsbury et al., Lancet 1: 1398–1402 (1987)). Similarly, overexpression of HER2 has also been correlated with other human carcinomas, including carcinoma of the stomach, endometrium, salivary gland, bladder, and lung (see, e.g. Yokota et al., Lancet 1: 765–67 (1986); Fukushigi et al., Mol. Cell. Biol. 6: 955–58 (1986)). Phosphorylation of such RTKs activates their cytoplasmic domain kinase function, which in turns activates downstream signaling molecules. RTKs are often phosphorylated at multiple different sites, such as distinct tyrosine residues. These enzymes are gaining popularity as potential drug targets for the treatment of cancer. For example, Iressa™, an inhibitor of EGFR, has recently entered clinical trials for the treatment of breast cancer.
FMS-related tyrosine kinase 3 (Flt3) is a receptor tyrosine kinase preferentially expressed in hematopoietic progenitor cells. The sequence for the human Flt3 gene has been published (see Small et al., Blood 15(4): 1110–9 (1993)). It has previously been shown that Flt3 is phosphorylated at tyrosine 958 in the C terminal domain (see Casteran et al., Cell Mol. Biol. 40(3): 443–56 (1994); Beslu et al., J. Biol. Chem. 271: 20075–81 (1996)). Recent studies have indicated that the Flt3 gene is mutated by internal tandem duplication in 20–25% of adults with acute myelogenous leukemia (AML), leading to phosphorylation and overactivation of Flt3 activity in cancerous cells (see Whitman et al., Cancer Res. 61(19): 7233–39 (2001) Kottardis et al., Blood 98(6): 1752–59 (2001)). AML is the most common type of leukemia in adults, with an estimated 10,000 new cases annually (source: The Leukemia & Lymphoma Society (2001)). Flt3 has also been implicated in neural-crest derived tumors and myelodysplastic syndromes (see Timeus et al., Lab Invest. 81(7): 1025–37 (2001); Zwierzina et al., Leukemia 13(4): 553–57 (1999)). Mutation of Flt3 at aspartic acid 835 (asp835) has been implicated in progression of AML (see Abu-Duhier et al., Br. J. Haematol. 113(4): 983–88 (2001)). Although patient risk of AML may be clinically detected by examining genetic mutation of the Flt3 gene, many diagnoses are not made until patients present with symptoms of the disease, such as easy bruising, anemia and fatigue, or low white cell count. In addition, activation of the Flt3 receptor kinase leading to AML may occur in the absence of genetic mutations of the Flt3 gene.
Inhibitors of Flt3 are presently being studied as potential AML therapeutics (see Naoe et al., Cancer Chemother. Pharmacol. 48: Suppl. 1: S27–30 (2001)). For example, agonist antibodies that bind the extracellular domain of Flt3 and activate its tyrosine kinase activity have been described (see U.S. Pat. No. 5,635,388, Bennett et al.). More recent results indicate that Flt3 inhibitors have anti-tumor activity in pre-clinical models (Weisberg et al., Cancer Cell 1(5): 433–43 (2002); Kelly et al., Cancer Cell 1(5): 421–32 (2002)). However, Flt3 expression alone does not always correlate with patient response (personal communication, Dr. Donald Small, Johns Hopkins University).
Accordingly, new and improved reagents for the detection of Flt3 activity would be desirable, including development of reagents against newly-identified sites of Flt3 phosphorylation. Since phosphorylation-dependent over-activation of Flt3 is associated with diseases such as AML, reagents enabling the specific detection of Flt3 activation would be useful tools for research and clinical applications.